Lactobacilli and Bifidobacterium as anti-atherosclerotic agents

Atherosclerosis is the thickening or hardening of the arteries which is caused by a buildup of atheromatous plaque in the inner lining of an artery. Hypercholesterolemia, inflammation, oxidative stress, and trimethylamine N-oxide (TMAO) are important risk factors for atherosclerosis. Therefore, this study aimed to review the anti-atherosclerotic effects of Lactobacilli and Bifidobacterium via improving lipid profile and reducing the effects of oxidative stress, inflammation, and TMAO. To prepare the present review, several databases such as Scopus, PubMed, and Google Scholar were searched, and relevant articles from 1990 until 2022 were selected and reviewed. The present review showed that Lactobacilli and Bifidobacterium reduce the risk of atherosclerosis in both in vitro and in vivo studies by breaking down or altering cholesterol metabolism with the help of their by-products and by reducing inflammation and oxidative stress and TMAO. Consumption of Lactobacilli and Bifidobacterium can be useful in prevention of atherosclerosis.


Introduction
Atherosclerosis is the thickening or hardening of the arteries that is caused by a buildup of atheromatous plaque in the inner lining of an artery. Atheromatous plaque is made up of deposits of fatty substances, cholesterol, cellular waste products, calcium, and fibrin (1). Atherosclerosis is a slow and progressive process that usually develops over several years through a complex series of cellular events occurring within the arterial wall and in response to a variety of local vascular circulating factors (2). Cardiovascular diseases (CVDs), which are predominantly the clinical manifestation of atherosclerosis, are the leading cause of death in all regions of the world except Africa (3). CVDs led to 17.3 million deaths in 2008 and it is estimated that by 2030, more than 23.3 million deaths will occur annually in the world (4). Coronary heart diseases (CHDs) are the most common cardiovascular diseases (CVDs) (5). CHDs burden costs $108.9, $23, £30, $94, and €196 billion per year in the United States, Canada, UK, Australia, and Europe, respectively (6)(7)(8). Hypercholesterolemia, inflammation and oxidative stress, and trimethylamine N-oxide (TMAO) are important risk factors for atherosclerosis (1,2,9).
Hypercholesterolemia is elevated levels of non-highdensity lipoprotein cholesterol (HDL-C) (subtraction of HDL-C from total cholesterol (TC)), particularly lowdensity lipoprotein cholesterol (LDL-C) (10). The low intake of high-fat diets, free sugar, and carbohydrates can reverse hypercholesterolemia and the risk of CVDs. The treatment of hypercholesterolemia is expensive with severe drawbacks (11). In the USA, about one-third of the hypercholesterolemic subjects are under control and only half of them can be treated (11). Studies have demonstrated that the risk of CHDs and CHDs-related mortality increase by about 35% and 45%, respectively for every 1 mmol increase in serum cholesterol levels; and the risk of CHDs decrease by 2%-3% for every 1% decrease in serum cholesterol levels (12).
The microbial flora of the gastrointestinal (GI) tract of humans and animals plays important roles in various functions. Recently, many scientists have been interested in the use of this useful microbial flora for sustaining and promoting human health (13).
Based on World Health Organization (WHO) definition, probiotics are "live microorganisms when administered in adequate amounts confer a health benefit on the host". These useful microorganisms are used in different forms such as yogurt, cheese, and fermented foods as well as capsules, powders, etc., as foods or therapeutic agents for health purposes (4,13).
Elie Metchnikoff is the first person that declared the useful benefits of probiotics. He believed that lactic acid bacteria (LAB), an important group of probiotics, contained in sour milk are the cause of the longevity of Bulgarian peasants and the inhibition of pathogens (13).
Lactobacilli and Bifidobacterium are the most well-known bacterial genera of probiotics which are generally regarded as safe (GRAS) and called friendly bacteria (14)(15)(16). It has been shown that several strains of Lactobacilli and Bifidobacterium are available for human use which have several health benefits, including improvement of intestinal health, amelioration of lactose intolerance symptoms, reduction of the risk of various other diseases such as infectious diseases and cancer (17,18). These microorganisms are able to reduce the risk of CVDs) blood which through LDL receptors transport cholesterol to a variety of tissues such as the wall of the artery which can promote atherosclerosis. In addition, LDL particles can directly bind to endothelial scavenger receptor class B type 1 (SR-B1) and penetrate to the subendothelial space of arteries then be oxidized, finally causing a buildup of immune cells and atherosclerotic plaque formation (31,32). Macrophages internalize oxidized LDL particles to form foam cells that often are trapped in the walls of arteries and contribute to forming atherosclerotic plaque. The atherosclerotic plaques are the main cause of CVD (32,33). HDL particles transport cholesterol from tissues to the liver, either for excretion or for other uses, in a process known as reverse cholesterol transport (RCT) (30).
The excess cholesterol in macrophages can also be excreted by the ATP-binding cassette subfamily A member 1 (ABCA1) or subfamily G member 1 (ABCG1) which prevents the formation of foam cells thereby reducing the risk of atherosclerosis and CVD (34).

Bile acids
Bile acids (BAs) are synthesized from cholesterol via the classical pathway or alternative pathway ( Figure 2). The classical pathway is the main production pathway of BAs (90%) in which cholesterol is converted to BAs by the ratelimiting enzyme cholesterol 7 α-hydroxylase (CYP7A1) in the liver. In the alternative pathway, the remaining 10% BAs are produced by sterol 27-hydrolase (CYP27A1) in extrahepatic sites including the vascular endothelium and macrophages (37). There are well-known feedback loops that control gene transcription of the key enzymes in BA synthesis. The nuclear farnesoid X receptor (FXR) is one of them that in an increased BA pool induces the expression of small heterodimer partner (SHP), thereby down-regulates CYP7A1expression (38). First, primary BAs, cholic acid, and chenodeoxycholic acid are synthesized and then conjugated with glycine or taurine residues to intestinal bile salts. Conjugated BAs are transported into bile by the bile salt export pump (BSEP) or by ABCG5/8 (39). These conjugated BAs are stored temporarily in the gallbladder until they are released into the small intestine upon meals to facilitate emulsion.
About 95% of bile acids are reabsorbed by active transport mainly via the apical sodium-dependent bile acid transporter (ASBT) from the ileum and secreted into the portal circulation by the organic solute transporters (OST α/β) and recycled back to the liver for further secretion into the biliary system and gallbladder. Increased levels of BAs in the ileum can activate FXR inhibiting ASBT and reabsorption of BAs. In addition, activation of FXR induces the production and secretion of fibroblast growth factor 19 (FGF19) which binds to fibroblast growth factor receptor 4 (FGFR4) and activates a signaling pathway that downregulates CYP7A1 (40).

Mechanisms for cholesterol reduction
There are several possible mechanisms for the cholesterollowering effect of Bifidobacterium and Lactobacilli which are obtained based on in vitro and animal studies ( Figure 3). These mechanisms include deconjugation of BAs by bile salt hydrolase (BSH), binding to cholesterol and incorporation into the cellular membrane (41,42), production of shortchain fatty acids (SCFAs) (33,43), trapping cholesterol by peptidoglycan and exopolysaccharides (44,45), and conversion of cholesterol to coprostanol (46).
The most profound cholesterol-lowering mechanism has been attributed to bile salt hydrolase (BSH) activity (41). Studies have shown that many Bifidobacterium and Lactobacilli species produce BSH which can cleave amide bonds of conjugated BAs and convert them to liberate free primary BAs and amino acid (taurine or glycine) in which BAs are more hydrophobic and less efficiently reabsorbed from intestines and thereby excreted in feces (41,(47)(48)(49). Therefore, BSH activity causes an increase in the production of BAs from cholesterol, thus leading to the reduction of cholesterol. Moreover, following the activity of BSH, due to low BAs, the formation of micelles is disrupted, thereby the cholesterol absorption in the intestine is decreased (50).
Another cholesterol-lowering mechanism of Lactobacilli and Bifidobacterium is the ability to bind cholesterol and incorporate it into cell membranes (51) The ability of cholesterol-binding appeared to be growth and strainspecific. It has been observed that, although live bacterial cells lower more cholesterol than dead cells, the dead bacterial cells can remove cholesterol from media, which indicates cholesterol can also be bound to the cellular surface (52). Some Lactobacilli species have been found to possess surface protease-sensitive receptors which bind to exogenous cholesterol or phosphatidylcholine vesicles and incorporate them into their cell membranes (42,53).  The cell wall amino acids and peptidoglycan as well as the Exo-polysaccharides (EPS) of probiotics have a role in the reduction of cholesterol (44,45). Cholesterol-lowering by this mechanism is however strain and growth-dependent (52). Some intestinal microorganisms such as Lactobacilli and Bifidobacterium species can synthesize different EPS (54)(55)(56). It has been observed that the amount of produced EPS is correlated with the quantity of assimilated cholesterol by probiotic strains. The EPS of Lactiplantibacillus plantarum BR2 and Lacticaseibacillus paracasei M7 showed a cholesterol-lowering effect (57).
Short-chain fatty acids (SCFAs) are important components produced from oligosaccharides by gut microbiota and also probiotic strains such as Lactobacilli and Bifidobacterium species which have been found to play a vital role in cholesterol reduction (43,58,59). It has been reported that SCFAs are able to activate peroxisome proliferator-activated receptors (PPARs). The peroxisome proliferator-activated receptor γ (PPARγ) controls angiopoietin-like protein 4 (ANGPTL4) which inhibits lipoprotein lipase (LPL) (60). Therefore, SCFAs suppress LPL activities by activation of PPARγ and up-regulating ANGPTL4 levels which leads to regulation of fatty acid oxidation in muscle and adipocytes that reduce fat storage (61,62). It has been reported that butyrate is able to inhibit the synthesis of cholesterol by reducing HMG-CoA reductase (HMGCR) activity which is the rate-limiting enzyme of cholesterol synthesis (63). SCFA can also stimulate the hepatic synthesis of BAs by up-regulation of CYP7A1 which leads to reduction of cholesterol (64). A high concentration of SCFA in the gut can lower colonic pH and promote the growth of Lactobacilli and Bifidobacterium species (54,65,66).
Another mechanism is the conversion of cholesterol into coprostanol which reduces cholesterol absorption and makes it easily eliminable with defection (67). The efficiency of this mechanism depends on the abundance of bacteria that possess reductase enzymes. This enzyme is present in some species of Lactobacilli and Bifidobacterium species such as L. acidophilus ATCC 314, L. acidophilus FTCC 0291, L. bulgaricus FTCC 0411, and Bifidobacterium bifidum PRL2010 (64). In another study, it has been observed that L. acidophilus, L. bulgaricus and Lacticaseibacillus casei ATCC 393 contain both intracellular and extracellular cholesterol reductase (20,42).
Liver X receptors (LXRs) can be stimulated by Lactobacilli and Bifidobacterium species which down-regulate NPC1L1 expression in the liver and intestine thereby reducing cholesterol absorption in the intestines and re-absorption in the liver (68)(69)(70). LXRs also down-regulate two genes, squalene synthase and lanosterol 14α-demethylase (CYP51A1) genes that encode key enzymes in the synthesis of cholesterol (71). In a study, L. plantarum lowered cholesterol levels via up-regulation of LXR (72,73). LXR also plays an important role in RCT by regulating several transporters including ABCA1 and ABCG1, so can help to reduce cholesterol and atherosclerosis (74). NPC1L1 acts as a negative regulator of NPC2, therefore down-regulation of NPC1L1 up-regulates NPC2 which in return increases cholesterol exporters such as ABCG5/8 (75).
Also, the farnesoid X receptor (FXR), a bile acid nuclear receptor may be stimulated by deconjugated BAs. FXR senses the increased level of hepatic BAs and induces the expression of SHP which down-regulates rate-limiting enzymes CYP7A1 and sterol-12α-hydroxylase (CYP8B1) (76,77).

In vitro and animal studies on cholesterol-lowering
Numerous studies have been performed on the cholesterollowering effect of Lactobacilli and Bifidobacterium species most of which are in vitro and animal experiments. These experiments have shown significant efficacy of Lactobacilli and Bifidobacterium species on cholesterol reduction (20,78,79).

Lactobacilli species
Lactobacilli species have been shown in various studies to have a high ability to decrease cholesterol (78,(80)(81)(82)(83)(84) some of which are listed below.
L. plantarum is the most common Lactobacilli investigated in cholesterol-lowering efficacy. In a study, the effect of L. plantarum PH04 at a dose of 10 7 colony-forming unit (CFU)/mouse daily for 14 days on serum cholesterol and TGs levels of hypercholesterolemic mice was investigated. L. plantarum PH04 resulted in 7% and 10% reduction in the serum cholesterol and TGs, respectively (82). Also, L. plantarum CK 102 at a dose of 5×10 7 CFU/ml has significantly reduced the serum levels of total cholesterol and LDL-C in Sprague-Dawley rats (85). In some studies, the cholesterol-lowering ability of L. plantarum in three conditions of growing, resting, and death has been examined. For example, in an in vitro study, four strains of L. plantarum (TGCM 15, TGCM 26, TGCM 33, and TGCM 128) showed more than 50%, (13.11-23.28%), and (11.44-19.53%) cholesterol reduction in growing, resting, and dead cells, respectively (83). In a similar study, L. plantarum strains isolated from mustard removed cholesterol by growing (110.67 to 167.03 μg/ml), resting (1.40 to 35.44 μg/ ml) and dead (0-54.21 μg/ml) cells (86). It has been shown that double-coated L. plantarum KCTC3928 with proteins and polysaccharides can significantly reduce the serum LDL-C and TGs levels in mice (87). Cholesterol-lowering ability of L. plantarum strains obtained from various sources such as kefir grains (47), traditional fermented milk (88), and local fermented food products (89) has been displayed. BSH activity and cholesterol coprecipitation are the most common mechanisms seen in L. plantarum strains (83,85). Expression of the LDL-R and HMGCR were marginally affected and CYP7A1 was significantly up-regulated following live L. plantarum KCTC3928 feeding (87,89).
Also, L. acidophilus has a remarkable cholesterollowering ability. In a study by Sarkar in 2003, the cholesterollowering ability of L. acidophilus was demonstrated. This study reported that cholesterol reduction is due to the assimilation or/and attachment of cholesterol to the surface of L. acidophilus cells (90). L. acidophilus also has shown BSH activity and co-precipitation with cholesterol ability, the ability that increases with decreasing pH (91). The cellfree supernatant (CFS) of L. acidophilus ATCC 43121 can reduce cholesterol in the broth in the presence of bile salts (92).
L. casei is another Lactobacilli with cholesterol-lowering ability. L. casei has demonstrated that it is able to reduce serum cholesterol levels alone (93) or/and in combination with prebiotics (94).
There are other Lactobacilli species that have been less examined for their cholesterol-lowering activities.
Lactobacillus crispatus isolated from gherkins (fermented cucumber) has significantly reduced cholesterol (95). Lactobacillus bulgaricus (96), Limosilactobacillus reuteri (97), and Lactobacillus gasseri SBT0270 (98) have significantly improved the lipid profile of rats, mice, and rats, respectively. In a study, L. plantarum 9-41-A and Limosilactobacillus fermentum M1-16 resulted in significant reduction in the serum levels of total cholesterol, LDL-C, and TGs in rats, whereas HDL-C level was not significantly changed (P>0.05) (99). In a similar study, the cholesterol-lowering effect of Lactobacillus pentosus KF923750 was examined on the lipid profile of rabbits. Total cholesterol, LDL-C, and TGs levels were significantly decreased, while no significant change was seen in HDL-C levels (100). Lacticaseibacillus rhamnosus BFE5264 could reduce the serum levels of cholesterol that were accompanied by changes in gut microbiota and production of SCFA (101

Bifidobacterium species
Bifidobacterium species have shown cholesterol-lowering ability in various studies (104)(105)(106)(107)(108)(109). Bifidobacterium longum (B. longum) and B. bifidum are the most common Bifidobacterium species that have been used to evaluate the cholesterol-lowering effects (48,(104)(105)(106)109). In a study, consumption of yogurt containing Bifidobacterium pseudocatenulatum G4 or B. longum BB536 for 8 weeks led to improvements in the lipid profile and increased fecal excretion of BAs in rats compared with the control groups (106). Administration of B. longum BB536 resulted in a significant reduction of total cholesterol, lipid deposition of liver and adipocyte size, and positively affected the function of kidney and liver in hypercholesterolaemic Sprague-Dawley rats. Cholesterol-lowering effect of B. longum BB536 significantly increased in the presence of inulin (105). Also, B. animalis subsp. Lactis F1-7 (77) and B. pseudolongum (107) have reduced the serum cholesterol levels in mice. It has been shown that the cholesterol-lowering efficacy of Bifidobacterium species increases in combination with Lactobacilli species (108). Ranji et al. (2019) reported that oral consumption of B. bifidum and Lactobacilli leads to a significant decrease in the TGs and LDL-C in mice (P<0.005) (104). In a study, B. longum strains (CCFM 1077, I3, J3, and B3) showed different levels of cholesterol reduction which raised the strain-specific effects in reducing cholesterol (109). BSH activity is the most common mechanism investigated in Bifidobacterium species (44,48).

Clinical studies on cholesterol-lowering
Several clinical studies have been performed on the cholesterol-lowering effects of Lactobacilli and Bifidobacterium species which are summarized in Table 1. Clinical studies are different based on country, bacteria, sample size, duration, dosage, delivery, subjects, and outcome.
Most clinical studies have been performed on hypercholesterolemic individuals. In two studies, Fuentes et al. evaluated the cholesterol-lowering effect of L. plantarum strains in hypercholesterolemic adults. They reported that administration of L. plantarum-containing capsules significantly improves the lipid profile of patients. They also declared that the biofunctionality of L. plantarum strains has better effects in patients with higher levels of cholesterol (110,111). Another study showed that administration of L. fermentum-containing capsules has no significant efficacy on serum lipids in individuals with elevated serum cholesterol (112). In Lewis et al. (2005) study, although L. acidophilus significantly reduced in vitro cholesterol, no effect was seen in the serum lipid profile of hypercholesterolaemic volunteers (113).
Also, a powder containing L. curvatus HY7601 and L. plantarum KY1032 has reduced TGs levels in hypertriglyceridemic individuals. (114). In addition to capsules and powder, other delivery methods have been used to evaluate the cholesterol-lowering efficacy of Lactobacilli and Bifidobacterium, especially in hypercholesterolemic individuals. Yogurt and fermented milk which contain Lactobacilli or/and Bifidobacterium are the most common delivery methods. It has been shown that probiotic milk formula (PMF) containing L. acidophilus (La5), L. casei (TMC), and B. lactis (Bb12) can significantly reduce the total cholesterol and LDL-C of hypercholesterolemic volunteers. (115). Also, milk fermented by B. longum strain BL1 (116) and fermented milk containing L. acidophilus l1 (117) have reduced the serum concentrations of total cholesterol, LDL-C, and TGs, while not changing the level of HDL-C. Combination of soy with a probiotic containing Lactobacillus acidophilus, B. bifidus, and Lactobacillus GG resulted in a significant reduction of total cholesterol and LDL-C in mildly hypercholesterolaemic subjects (118). It has been found that in addition to in vitro and animal studies, BSHactive Lactobacilli and Bifidobacterium have remarkable effects on cholesterol in human studies. L. plantarum ECGC 13110402 (119) and L. reuteri NCIMB 30242 (120), strains with high BSH activity, have demonstrated significant improvement in the lipid profile of hypercholesterolaemic subjects.
After hypercholesterolemic subjects, most clinical studies have been done on the lipid profile of healthy subjects. It seems that Lactobacilli and Bifidobacterium are more effective in hypercholesterolemic subjects than in healthy subjects. Although yogurt containing L. acidophilus La5 and B. lactis Bb12 (121) and probiotic yogurt fermented with L. acidophilus and B. lactis (122) have decreased total cholesterol, they had no significant effects on TGs and LDL-C in healthy adults. Also, in other studies, B. animalis subsp. lactis BB-12 and L. acidophilus La5 did not significantly improve the lipid profile including total cholesterol, LDL-C, HDL-C, and TGs in healthy participants (123,124).
In type 2 diabetic patients, consumption of probiotic yogurt containing L. acidophilus and B. lactis resulted in a 4.54% decrease in total cholesterol and a 7.45% in LDL-C, and a significant reduction in the total cholesterol:HDL-C ratio and LDL-C:HDL-C ratio (125), but consumption of probiotic-containing lactobacilli did not reduce the serum TGs concentration, total cholesterol, and LDL-C levels (126).
It has been shown that L. acidophilus DDS-1, B. longum, and L. plantarum 299v do not change significantly the serum cholesterol levels in postmenopausal women (127) and smokers (128), respectively.
Although improved lipid profiles are often associated with decreased serum levels of LDL-C and total cholesterol and increased HDL-C, in many clinical studies many cases were unchanged in HDL-C. Even in a study by Rerksuppaphol et al. (2015) (129), HDL-C decreased which seems illogical, and the author did not explain it.

Effects on inflammation and oxidative stress and trimethylamine-N-oxide (TMAO)
Hypercholesterolemia has been generally considered an important cause of atherosclerosis (130). However, other studies have demonstrated that despite the reduction of serum level of LDL-C more than 50% of cardiovascular risk still remains (131,132). So, we have to look for other causes of this important disease, one of which is inflammation and oxidative stress and the other is trimethylamine N-oxide (TMAO). There are studies that have shown the beneficial effects of probiotics on these atherogenic factors (21,22). Figure 4 shows the hypothesized anti-atherosclerotic mechanisms of Lactobacilli and Bifidobacterium on inflammation and oxidative stress and TMAO based on performed studies.

Inflammatory and oxidative stress factors
Studies have shown that in addition to hypercholesterolemia, inflammation and oxidative stress also play a vital role in the progression of atherosclerosis (21). Atherosclerosis is regulated by various immune cells such as macrophages, lymphocytes, and dendritic cells, and causes foam cells to form in the subendothelial region by the deposition of cholesterol. Atherosclerotic plaques express a complex network of proinflammatory cytokines, including interleukins (IL), tumor necrosis factor-alpha (TNF-α), and interferons (21,133). IL-1β has substantial  (21). TNF-α produced by smooth muscle cells, macrophages, and endothelial cells of atherosclerotic arteries, has potent estrogenic effects via stimulating the formation of new vessels, adhesion of leukocytes to endothelial cells, chemotaxis, and developing atheroma features. In addition, inhibitors of nuclear factor kappa B (IκB) proteins including IκBα, β, and γ are regulatory proteins that bind to nuclear factor kappa B (NF-κB) and prevent transcription and nuclear translocation of NF-κB, a factor with an important role in the expression of proinflammatory cytokines.
Oxidative stress is the overproduction of reactive oxygen species (ROS) in such a way that anti-oxidant systems are unable to control or repair their damage (134). ROS including free oxygen radicals, oxygen ions, and peroxides at moderate concentrations play critical roles in the regulation of various cell functions and biological processes such as vascular tone, oxygen sensing, cell growth and proliferation, apoptosis, and inflammatory responses (135). Uncontrolled production of ROS resulted in oxidative stress and endothelial dysfunction. Oxidative stress plays an important role in the atherosclerosis process (135).
Malondialdehyde (MDA) and oxidized low-density lipoprotein (oxLDL) are oxidative biomarkers that arise following oxidative stress and lipid peroxidation by ROS (134,136). It has been shown that ox-LDL plays a key role in several steps of atherogenesis and also inhibits endothelial nitric oxide synthase (eNOS) activity in endothelial cells (134,137). In several studies, ox-LDL was elevated in the plasma of atherosclerotic patients (134,138). Superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), paraoxonase (PON), and thioredoxins are major antioxidants in the vascular wall (4,134).
Nitric oxide (NO) synthesized by endothelial nitric oxide synthase (eNOS) and/or neuronal nitric oxide synthase (nNOS) have anti-atherosclerotic activity, whereas NO produced by inducible nitric oxide synthase (iNOS) plays a pro-atherogenic role (135). Activation of iNOS can form uncoupled eNOS which is a ROS generator and contributes to atherogenesis (134). Inflammation, oxidative stress, bacterial endotoxins, and sepsis, activate iNOS, which produces Ca 2+ -independent NO at a rate 1,000-fold greater than that of eNOS (137,139).
Various studies have demonstrated the ability of Lactobacilli and Bifidobacterium species in modulating inflammation and oxidative stress (140)(141)(142)(143). In a study, Hassan et al. (2020) reported that L. plantarum ATCC 14917 is able to exert its anti-atherosclerotic effects via modulation of proinflammatory cytokines and oxidative stress. They observed that feeding L. plantarum ATCC 14917 to Apo E−/− mice daily for 12 weeks had no effect on body weight and lipid profile, but significantly inhibited atherosclerotic lesion formation. In addition, the oxidative stress factors such as oxLDL and MDA, as well as inflammatory factors including NF-κB and IL-1β levels were significantly reduced, whereas the level of SOD was induced. Furthermore, administration of L. plantarum ATCC 14917 significantly attenuated the IκBα protein degradation and inhibited translocation of the p65 subunit of NF-κB, and also modulated the gut microbiota composition in ApoE−/− mice (21). In another study, Fang et al. (2019) evaluated the effect of L. rhamnosus GR-1 on atherosclerotic plaque formation in ApoE-/-mice fed with a high-fat diet.
Administration of GR-1 had no efficacy in body weight or serum levels of lipid, but reduced the progression of atherosclerosis and plaque formation. Also, administration of GR-1 reduced the values of oxLDL and MDA, inhibited the translocation of NF-κB p65, and diminished the expression of inflammation cytokines TNF-α and IL-6 (132). Also, Chen et al. (2013) showed similar results when using L. acidophilus ATCC 4356 (142). Treatment with L. fermentum MTCC: 5898 had beneficial effects on inflammation and oxidative stress in hypercholesterolemic Wistar rats. It significantly reduced atherogenic index, coronary artery risk index, lipid peroxidation, and mRNA expression of inflammatory cytokines (TNF-α and IL-6) and significantly increased anti-oxidative enzymes (catalase, SOD, and GSH-Px) (144). In another study, the anti-oxidative effects of L. casei Zhang on hyperlipidemic rats were evaluated. Treatment with L. casei Zhang significantly decreased MDA levels and increased SOD and GSH-Px in the serum and liver of rats. Authors also declared that the anti-oxidative effect of L. casei Zhang is more effective in hyperlipidemic rats compared with normal rats, and application of different therapeutics doses have variant effects on hyperlipidemic rats (145). Kim et al. (2013) examined the inhibitory effects of (pLTA)-lipoteichoic acid (LTA) of L. plantarum-on atherosclerotic inflammation. They observed that pLTA prevented the production of proinflammatory cytokines and NO in lipopolysaccharide (LPS)-stimulated cells. The authors declared that the inhibitory effect of pLTA is due to inhibition of NF-κB and activation of mitogen-activated protein kinases (MAP kinases). They concluded that their results emphasize the role of pLTA in suppressing atherosclerotic inflammation (146).

Trimethylamine-N-oxide (TMAO)
It has been shown that one of the causes of atherosclerosis and cardiovascular diseases is linked to the high level of TMAO (147). TMAO is oxidized from trimethylamine (TMA) by hepatic flavin-containing monooxygenase3 (FMO3). TMA is produced from dietary L-carnitine, phosphatidylcholine, choline, and betaine by intestinal microbiota (22). Precursors of TMA are highly present in foods of animal origin such as red meat, dairy products, and eggs. Seafood such as fish has more carnitine and choline and also TMA and TMAO compared with other foods (148). The serum level of TMAO is dictated by several factors such as gut microbiota, diet, drug administration, and liver FMO3 activity (149). TMAO mediated by intestinal microbiota has been a promising target for the treatment of atherosclerosis (150).
Although the atherosclerotic mechanisms of TMAO are not fully understood, some hypotheses are related to cholesterol metabolism. TMAO can activate SHP and FXR and down-regulate enzymes that synthesize BAs including CYP7A1 and CYP27A1 (151). Activation of FXR can decrease reverse cholesterol transport and induce FMO3 which can lead to atherosclerosis. It has been shown that TMAO is able to reduce the expression of NPC1L1 and ABCG5/8 (152) and also up-regulate ABCG5/8 in the small intestine (153). Some studies have shown the down-regulation effect of TMAO on ABCA1 and ABCG1 (152)(153)(154) and scavenger receptor A (SRA) and cluster of differentiation 36 (CD36) (155) expression in macrophages which cause the formation of foam cells and atherosclerotic plaques. It has been suggested that supplementation with probiotic strains such as Lactobacilli and Bifidobacterium strains might change TMAO levels. Qiu et al. (2018) showed that L. plantarum ZDY04 significantly reduced serum TMAO and cecal TMA levels by modulating the gut microbiota in apolipoprotein-E knockout (ApoE−/−) mice (a mouse model of atherosclerosis). They also reported that the reduction of serum TMAO and cecal TMA was not due to influencing the expression levels of hepatic FMO3 and metabolizing choline, TMA, and TMAO. In this study, L. plantarum ZDY04 could significantly inhibit the development of TMAO-induced atherosclerosis in ApoE−/− mice as compared with the control (22).

Discussion
Here we reviewed the effects of Lactobacilli and Bifidobacterium on important risk factors of atherosclerosis including hypercholesterolemia, inflammation, oxidative stress, and TMAO. Given that hypercholesterolemia is one of the most important risk factors for atherosclerosis, a significant part of the present review is devoted to the cholesterol-lowering effects of these probiotics in vitro, animal, and human studies. This review showed that in vitro and animal studies have shown a greater cholesterol-lowering effect than human studies. The reasons for these differences may include differences in bacterial species and strains, duration of the studies, dosage, delivery method, studied individuals, and also the used composition. The present review mentioned various possible cholesterol-lowering mechanisms of Lactobacilli and Bifidobacterium, of which, deconjugation of bile by the BSH enzyme is more studied and well-known. In reviewed studies, Lactobacilli and Bifidobacterium strains that have shown high BSH activity significantly reduced the cholesterol levels. This mechanism is not yet clear enough, so further studies are needed to determine the exact effects of the BSH enzyme. In reviewed clinical studies, the cholesterol-lowering ability of probiotic strains in hypercholesterolemic individuals was more promising than in other individuals. It can be found out that although several clinical studies have been performed on cholesterol reduction by Lactobacilli and Bifidobacterium strains, most of them have only examined their effectiveness, and unlike in vitro and animal studies, the mechanism of cholesterol reduction has not been studied. Therefore, more studies are needed in both in vitro and in vivo (animal and human) conditions to shed more light on the effects and mechanisms of cholesterol-lowering. However, other studies have shown that despite reduced serum LDL-C levels, more than 50% of cardiovascular risks persist which raises the involvement of other factors in causing this disease. Indeed, inflammation and oxidative stress are now widely considered to play a vital role in atherosclerotic progression. Thus, in addition to the cholesterol-lowering effect, the effects of Lactobacilli and Bifidobacterium on inflammation and oxidative stress have attracted the attention of researchers and fortunately yielded promising results. An important point in our review is that although some Lactobacilli and Bifidobacterium strains may not show significant cholesterol-lowering effects in human studies, it should be hoped that these strains can have anti-atherosclerotic effects via reduction of inflammation, oxidative stress, and TMAO.

Conclusion
This review presented the effects of two well-known bacterial probiotics including Lactobacilli and Bifidobacterium on the most important risk factors for atherosclerosis such as hypercholesterolemia, inflammation, oxidative stress, and TMAO. Our review showed that these microorganisms are able to reduce cholesterol significantly in vitro, in animals and humans, although this ability is greater in vitro in animals than in humans. These useful microorganisms are also able to reduce the risk of atherosclerosis via reducing the serum levels of TMAO by improving the gut microbiota. Also, it can be concluded that these probiotics can diminish the risk of atherosclerosis by inhibiting inflammation and oxidative stress, an interesting and promising mechanism. Finally, we propose further studies to elucidate the effects and mechanisms of cholesterol-lowering, serum TMAO reduction, and inhibition of inflammation and oxidative stress by Lactobacilli and Bifidobacterium.